Isolated nucleic acids, particularly those that encode proteins, are attractive candidates for a variety of clinical applications. In particular RNAs that encode proteins, such as mRNAs, provide a number of potential advantages for clinical applications. For example, RNA can be transfected into cell in vivo, in vitro or ex vivo to induce expression of desired therapeutic or diagnostic proteins for treating or diagnosing disease. However, the potential of RNA therapeutics is limited by stability and half-life of the RNA as well as by the level of expression of the encoded protein.
Naturally occurring mRNAs contain a 5′ cap structure which helps stabilize the RNA and is fundamental to eukaryotic gene expression (Shuman, S. et al Mol. Microbiol. 1995, 17, 405-410). These mRNAs also contain a 3′ poly A tail. Both modification stabilize and improve translation of the encoded protein. The 5′-cap structure found at the 5′ end of eukaryotic messenger RNAs (mRNAs) and many viral RNAs consists of a N7-methylguanosine nucleoside linked to the 5′-terminal nucleoside of the pre-mRNA via a 5′-5′ triphosphate linkage (Shatkin, A. J. Cell 1976, 2, 645-53; Shuman, S. Prog. Nucleic Acid Res. Mol. Biol. 2001, 66, 1-40; Decroly, E. et al PLoS Pathog. 2011, 7, e1002059). This cap structure fulfills many roles that ultimately lead to mRNA translation. RNA capping is also important for other processes, such as RNA splicing and export from the nucleus and to avoid recognition of mRNA by the cellular innate immunity machinery (Daffis, S. et al Nature 2010, 468, 452-6; Zist, R. et al Nat. Immunol. 2011, 12, 137-43). A number of synthetic cap analogs have been described. See, e.g., WO2009/149253, WO2011/015347.
Synthetic mRNAs are typically prepared by enzymatic synthesis using RNA polymerase and a DNA template followed by enzymatic addition of the 5′-cap and the 3′-end (Peyrane, F. et al Nucleic Acids Res. 2007, 35, e26). However, the process is expensive and difficult to control and therefore undesirable for commercial scale production.
Thus, a need exists for RNAs that are modified at the 5′-end, the 3′-end or the 5′-end and 3′-end that can be efficiently produced and that have improved expression of products (e.g., protein) encoded by the RNA, lower immunogenicity and/or improve stability.